Oral Carcinogenesis--Human Gingival Kerocytes

正常人牙龈角质形成细胞（NHGK）在高Ca++培养基中以与体内类似的方式达到汇合后经历终末分化。诱导TGase 1活性（5至10倍增加）后形成不溶性细胞包膜（CE），表明TGase 1是口腔角质形成细胞终末分化的关键标志物。在终末分化的NHGK细胞中，其他终末分化标志物，例如外皮蛋白、SPR 1和膜联蛋白1的mRNA水平也增加。SPR 1、膜联蛋白1、兜甲蛋白、膜斑蛋白、桥粒斑蛋白、外皮蛋白、半胱氨酸蛋白酶抑制剂α和泛角蛋白被鉴定为NHGK CE的组分。这些CE含有异常高的量的SPR 1，表明SPR 1在口腔上皮的专门屏障功能中的重要作用。我们比较了细胞角蛋白1、5、8、10、14和19以及TGase I和TGase II在NHGK细胞和几种头颈鳞状细胞癌细胞系包括HN 4、HN 12、HN 8、HN 22、HN 30、HN 31、HN 13和HN 19中的表达。在大多数HNSCC系中，主要细胞角蛋白（1、5、10和14）的表达显著降低。部分癌细胞角蛋白8或19表达异常。与NHGK细胞相反，HNSCC细胞不能诱导TGase 1，也不能形成皮质细胞包膜，这与它们丧失终末分化能力一致。TGase 2是不可诱导的，但在HN 12、HN 30和HN 31细胞中观察到异常高水平的TGase 2。这些HN细胞的TGase 2活性与裸鼠成瘤性无关。永生化的人牙龈角质形成细胞（IHGK）显示出与NHGK相似的细胞角蛋白表达模式。与NHGK细胞一样，用编码HPV 16 E6/E7基因的pBabe载体永生化的IHGK细胞在高Ca++培养基中达到汇合后后经历终末分化。为了了解口腔癌发生的遗传和分子机制，我们使用含有6720个cDNA的cDNA微阵列（NCI-OncoChip）在NHGK、IHGK、HSG（人唾液腺系）、SGT（人唾液腺癌系）和11个HNSCC系中进行了基因表达谱分析。S.分析正在进行中，以将基因表达模式与细胞的表型相关联，并确定口腔癌发生中涉及的遗传变化。

Normal human gingival keratinocyte (NHGK) cells undergo terminal differentiation upon reaching confluency in high Ca++ medium in a similar fashion as in vivo. Induction of TGase 1 activity (5 to 10-fold increase) was followed by formation of insoluble cell envelopes (CEs) suggesting that TGase 1 is a key marker for terminal differentiation of oral keratinocytes. The mRNA levels of other markers of terminal differentiation, e.g. involucrin, SPR1 and annexin 1, were also increased in the terminally differentiating NHGK cells. SPR1, annexin 1, loricrin, envoplakin, desmoplakin, involucrin, cystatin alpha and pancornulin were identified as components of NHGK CEs. These CEs contained an unusually high amount of SPR1, suggesting an important role of SPR1 in the specialized barrier function of oral epithelium. We have compared the expression of cytokeratins 1, 5, 8, 10, 14 and 19, TGase I and TGase II in NHGK cells and several HNSCC (head and neck squamous cell carcinoma) lines including HN4, HN12, HN8, HN22, HN30, HN31, HN13 and HN19. The expression of the major cytokeratins (1, 5, 10 and 14) was significantly reduced in most HNSCC lines. Aberrant expression of cytokeratin 8 or 19 was observed in some carcinoma cells. In contrast to NHGK cells, HNSCC cells failed to induce TGase 1 and failed to form cornified cell envelopes, consistent with their loss of capacity for terminal differentiation. TGase 2 was not inducible but an unusually high level of TGase 2 was observed in HN12, HN30 and HN31 cells. The TGase 2 activity of these HN cells did not correlate with tumorigenicity in nude mice. The immortalized human gingival keratinocytes (IHGK) show a similar pattern of cytokeratin expression, as do NHGK. Like NHGK cells, IHGK cells, immortalized with pBabe vector encoding HPV 16 E6/E7 genes, undergo terminal differentiation upon reaching post-confluency in a high Ca++ medium. In an effort to understand the genetic and molecular mechanisms involved in oral carcinogenesis, we have conducted gene expression profiling in NHGK, IHGK, HSG (human salivary gland line), SGT (human salivary carcinoma line) and 11 HNSCC lines using cDNA microarrays (NCI-OncoChip) containing 6720 cDNA?s. Analysis is underway to correlate the gene expression patterns with the phenotypes of the cells and to identify genetic changes involved in oral carcinogenesis.